Extracellular enzyme activity of entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae and their pathogenicity potential as a bio-control agent against whitefly pests, Bemisia tabaci and Trialeurodes vaporariorum (Hemiptera: Aleyrodidae)

Objective This study was aimed to assess the enzymatic activity and pathogenicity potential of Beauveria bassiana and Metarhizium anisopliae against whiteflies in Ethiopia. Results The data showed that Beauveria bassiana AAUMB-29, AAUMFB-77, and AAUEB-59 generated the highest chitinase (EI = 3.41), lipase (EI = 4.45), and protease activities (EI = 5.44) respectively. The pathogenicity study of isolates on whitefly nymphs and adults indicated significant variation (P < 0.05) with mortality ranging from 71.67 to 98.33% and 60 to 100% against Bemisia tabaci and Trialeurodes vaporariorum nymphs respectively. The mortality of adults was between 58 and 94.27% against B. tabaci and 59.03 to 95.37% against T.vaporariorum. The result also showed that AAUMB-29, AAUMFB-77, and AAUDM-43 were the most virulent with LC50 values of 2.7 × 104, 5.3 × 104, and 5.4 × 104 conidia/ml against nymphs of B. tabaci, and with LC50 values 6.8 × 104, 8.2 × 104, and 7.2 × 104 conidia/ml against nymphs of T. vaporariorum, respectively. The B. bassiana AAUMB-29, B. bassiana AAUMFB-77, and M. anisopliae AAUDM-43 induced the highest whitefly mortality than other isolates. These isolates can be recommended for further tests under field conditions to fully realize their potential as effective biocontrol agents against whitefly pests in tomato. Supplementary Information The online version contains supplementary material available at 10.1186/s13104-022-06004-4.


Introduction
Whiteflies Bemisia tabaci and Trialeurodes vaporariorum (Hemiptera: Aleyrodidae) are notorious sap-sucking pests causing serious damage to vegetable crops through direct feeding and transmission of several plant viruses [1]. Tomato (Solanum lycopersicum) is one of the most favorable hosts of B. tabaci and T. vaporariorum [2], and these whitefly species cause tomato yield losses from 50 to 100% [3]. The control of whitefly is mainly dependent upon chemical pesticides to reduce agronomic losses [4,5]. However, the use of chemical pesticides induces pest resistance and an outbreak of secondary pathogens [6].
Entomopathogenic fungi (EPF) Beauveria bassiana and Metarhizium anisopliae are the most environmentallyfriendly bio-control agents against sucking insect pests [7]. They produce adhesion factors, cuticle degrading enzymes, infection structures [8], and toxic secondary metabolites to overcome host cuticles and cause infection [9]. These typical features of B. bassiana and M. anisopliae provide an advantage to effectively control sap-sucking insect species over others [10]. Different laboratory and field studies demonstrated that isolates of B. bassiana and M. anisopliae effectively controlled B. tabaci and T. vaporariorum with mortality ranging from 71 to 96.61% [11][12][13].
Increased interests in the use of entomopathogenic fungi in pest management options necessitate the selection of fungal isolates with high virulence that shows significant enzyme activities on target insects. In Ethiopia, locally isolated entomopathogenic fungi showed promising results for the control of agricultural pests such as Aphis gossypiin [14], Pachnoda interrupta [15], and Tuta absoluta [16]. However, there is a limited report on the use of these native isolates for the management of whiteflies. Hence, this study was carried out to evaluate the enzymatic activity and pathogenicity competence of B. bassiana and M. anisopliae against whitefly species, B. tabaci and T. vaporariorum in Ethiopia.

Material and methods
Indigenous entomopathogenic fungi B. bassiana and M. anisopliae were used in trials (Additional file 1: Table S1). Isolates were obtained from soil samples collected from farmlands and forest sites of Ethiopia. The potential isolates were selected based on their virulence effectiveness [17].

Pathogenicity test against whitefly nymphs and adults
Adult and nymph whiteflies were released to tomato leaves containing sprayed residues of fungal isolates as described by Mascarin et al. [22]. Tomato leaves were sprayed with 3 ml of a conidial suspension of isolates at 1 × 10 7 conidia/ml. After spraying, leaves were placed onto 0.2% water agar in a petri dish and adult whiteflies were released into treated leaves (15 adults/leaf ) in triplicates and incubated at 25 °C for 10 days. Similarly, the mortality of whitefly nymphs was assessed by spraying leaf discs (30 mm in diameter) containing 20 nymphs with 3 ml of conidial suspension of 1 × 10 7 conidia/ml. Then leaf discs were placed onto 0.2% water agar medium in a Petri dish and incubated at 25 °C for 10 days. The median of lethal time (LT 50 ) of each isolates at 10 days of post inoculation was calculated using probit analysis.

Sporulation of isolates on whitefly nymph cadavers
The spore production of isolates was assessed according to Mascarin et al. [22]. To quantify yield, four sporulated nymphs were randomly selected within each treatment and transferred into a 1.8 ml microcentrifuge tube containing 1.5 ml of 0.1% Triton X-100 and from which 1 ml was counted in triplicates using a hemocytometer.

Multiple-dose responses studies
The multiple-dose bioassay (1 × 10 5 -1 × 10 8 conidia/ml) was evaluated to estimate the average lethal concentration (LC 50 ) values of each isolate [23]. Each treatment was undertaken in triplicates to record nymph mortality for 10 days with periodic observation every day.

Data analysis
Mortality data were corrected using Abbott's formula [24]. The corrected mortality and spore per whitefly Enzymatic Index (EI) = Hydrolysis zone diameter Colony growth diameter .
nymph cadaver were arcsine transformed [25] and subjected to the ANOVA procedure in SPSS version 20. The bioassay evaluation was tested by means separated using Tukey's HSD test at P < 0.05. The lethal time (LT 50 ) and the lethal concentration (LC 50 and LC 90 ) values were determined with probit analysis (IBM SPSS statics 20) [26].

Cuticle degrading enzymatic activities
Entomopathogenic fungi were showed significant differences in their relative enzyme activities ranging from 1.20 to 3.41 for chitinase, 1.58 to 4.45 for lipase, and 1.72 to 5.44 for protease (Table 1). On average, the isolates displayed the highest protease index (3.41), followed by the lipase index (2.86) and chitinase index (2.42), respectively. Isolates showed significant differences in their enzyme activities; where almost all isolates (92%) showed excellent protease activities while 75% and 50% of the isolates displayed excellent lipase and chitinase activities respectively (Additional file 1: Fig. S1). Although 67% of the isolates showed excellent overall activities, B. bassiana AAUMB-29 performed best in chitinase activity (EI = 3.41), whereas B. bassiana AAUMFB-77 and B. bassiana AAUEB-59 exhibited the highest lipase (EI = 4.45), and protease activity (EI = 5.44) respectively.

Virulence of B. bassiana and M. anisopliae isolates against whitefly adults
All isolates were pathogenic to whiteflies, B. tabaci, and T. vaporariorum adults (Fig. 1)

Discussion
The main important bio-insecticidal traits of entomopathogenic fungi are the production of cuticle degrading extracellular enzymes [27]. Consequently, isolates of B. bassiana, and M. anisopliae were produced chitinase, lipase, and protease enzymes. The data showed that isolates differed in their chitinase, lipase, and protease enzyme activities that are attributed to their intraspecific and interspecific variability [28]. The three potential isolates, B. bassiana AAUMB-29, B. bassiana AAUMFB-77, and M. anisopliae AAUDM-43 displayed a high level of chitinase, lipase, and protease activities. Hence, the greater chitinase, lipase, and protease activities of isolates indicate the capability of protein, chitin, and lipids breakdown by these isolates. This alludes to that the fungal isolates are capable of successful penetration of insect cuticles [29,30], with a high virulence effect against target insects [31].
In this study, the M. anisopliae AAUDM-43 induced the highest mortality of 94.27% on B. tabaci whereas B. bassiana AAUMFB-77 inflicted greater mortality of 95.37% on the T. vaporariorum adults after 10 days of treatment at the rate of 1 × 10 7 conidia/ml. Among the